This invention relates to adjuvant compositions for stimulating an immune response to an antigenic substance when co-administered to an animal with said antigenic substance, and to vaccines containing said adjuvant composition.
Vaccination against disease has a long history. In general terms the technique involves injection of an antigenic substance, or antigen, into an animal whereby the presence of the antigenic substance generates an immune response in the animal. Classical vaccination techniques involve the injection of killed organisms but more recently vaccines comprising attenuated live organisms or antigenic components of an organism have been developed. It is frequently found with killed vaccines and, more particularly, with vaccines comprising a component of an organism that the immune response is substantially less than the response to natural infection. However, the effectiveness of such vaccines can be considerably enhanced by the co-administration of a suitable adjuvant composition with the antigenic substance. Adjuvants, while not necessarily being antigenic themselves, potentiate or enhance an animal""s immune response to the antigenic substance with which it is challenged. There are many adjuvants known and used but there is an ongoing need to identify new and effective adjuvants which are inexpensive, which produce minimal injection site irritation and discomfort and which are widely applicable and effective.
A common formulation for vaccines is to present the antigen(s) in an aluminium hydroxide gel. While this is effective in some cases and is reasonably benign, in many cases this adjuvant fails to induce a sufficiently protective response. It is also well known that antigens emulsified in a mineral oil vehicle together with whole mycobacterial cells (Freund""s complete adjuvant, FCA) can produce a generally effective immune response against a wide range of antigens. However, this formulation is unacceptable for routine use because of the inflammation, granulomas, ulceration and other lesions which can be formed at the injection site. Mineral oils alone (frequently referred to as Freund""s Incomplete Adjuvant, FIA or Incomplete Freund""s, ICF) are less damaging but are also less effective. Neutral oils (such as miglyol) and vegetable oils (such as arachis oil), ISCOMS and liposomes have also been used. Also effective are adjuvants containing purified mycobacterial component such as N-acetylmuramyl-L-alanyl-D-isogulutamine (MDP) or its analogues in aqueous or oil formulations. Among other adjuvants which have been or are currently used are the saponins, particularly triterpenoid mixtures such as Quil A (a purified extract from the bark of the tree Quillaja saponarioa) in aqueous solution or in the form of a matrix with cholesterol. Polycations such as diethylaminoethyldextran (DEAE dextran) can also be effective as adjuvants in some cases.
There have also been proposals to use a combination of two adjuvants substances in an adjuvant composition. For example, Australian patent no. 602348 describes an immunoadjuvant comprising an immunoadjuvant oil substantially free of mycobacteria and a polycationic polyelectrolyte immunoadjuvant such as DEAE dextran in the form of an emulsion having the polycationic polyelectrolyte dissolved in the aqueous phase. The two-component immunoadjuvant is said to overcome the rapid decline in the immune response associated with polycationic polyelectrolyte adjuvants on the one hand and, on the other, the weak initial response associated with immunoadjuvant oils. Accordingly, the two-component adjuvant is said to fill the gap in the prior art between those adjuvants inducing high peak/short life antibody responses and those inducing low peak/long life responses.
International application no. 88/07547 is primarily concerned with a novel peptide nevertheless, it also discloses the use of a novel adjuvant comprising DEAE dextran and a saponin or aluminium hydroxide and notes an improved antibody titre when the two-component immunoadjuvants are used. In particular, solutions of DEAE dextran and saponin in phosphate buffered saline are used but there is no suggestion of the incorporation of an immunoadjavent oil into such compositions.
Australian patent no. 640414 discloses a solid vaccine composition comprising an antigenic substance capable of inducing the generation of antibodies on parenteral administration to an animal, a saponin and a polycationic adjuvant. The essence of the invention is that the vaccine is formulated as solid to be implanted in the animal to thereby induce a long-lasting immune response. There is no suggestion of the presence of an immunoadjuvants oil in the composition and, indeed, the specification teaches away from the use of an oil as it is critical to the invention that this formulation be solid.
In the present invention it has been found, surprisingly, that combinations of certain adjuvants enhance the effectiveness of an antigenic substance in stimulating an immune response to a much greater extent than the sum of the profiles that would be obtained by the use of the components separately or through the use of a two-component immunoadjuvant.
According to a first aspect of the present invention there is provided an adjuvant composition for stimulating an effective immune response in an animal to an antigenic substance when co-administered to said animal with said antigenic substance, comprising:
(a) a saponin with immune stimulating activity;
(b) a polycationic polyelectrolyte with immune stimulating activity; and
(c) an immunoadjuvant oil.
According to a second aspect of the present invention there is provided a vaccine for administration to an animal, comprising:
(1) an antigenic substance; and
(2) an adjuvant composition comprising:
(a) a saponin with immune stimulating activity;
(b) a polycationic polyelectrolyte with immune stimulating activity;
(c) an immunoadjuvant oil.
According to a third aspect of the present invention there is provided a method of stimulating an effective immune response in an animal to an antigenic substance, comprising the steps of:
(1) providing said antigenic substance;
(2) providing an adjuvant composition for stimulating an effective immune response to said antigenic substance, comprising:
(a) a saponin with immune stimulating activity;
(b) a polycationic polyelectrolyte with immune stimulating activity; and
(c) an immunoadjuvant oil; and
(3) challenging said animal with said antigenic substance and said adjuvant composition.
According to a fourth aspect of the present invention there is provided the use of an adjuvant composition comprising:
(a) a saponin with immune stimulating activity;
(b) a polycationic polyelectrolyte with immune stimulating activity; and
(c) an immunoadjuvant oil to stimulate an effective immune response in an animal challenged with an antigenic substance.
According to a fifth aspect of the present invention there is provided the use of an adjuvant composition comprising:
(a) a saponin with immune stimulating activity;
(b) a polycationic polyelectrolyte with immune stimulating activity; and
(c) an immunoadjuvant oil in the preparation of a medicament for administration to an animal, wherein said medicament further comprises an antigenic substance.
The saponins are common secondary constituents of plants and typically are glycosides composed of several (hydrophilic) sugars in association with a (hydrophobic) molecule, which can be either a steroid or triterpenoid structure. In particular, an extract from the South American tree Quillaja saponarioa shows good adjuvant activity and is now denoted xe2x80x9cQuil Axe2x80x9d. While the precise chemical composition of Quil A is not known, the sugar moieties detected in the mixture include rhamnose, fucose, arabinose, xylose, galactose, glucose, apiose and glucuronic acid and the hydrophobic moiety has a triterpenoid structure. The nature of Quil A is discussed as length in Australian patent application no. 10777/95, the disclosure of which is incorporated herein by reference.
Preferably, the saponin is a triterpenoid compound or a mixture of triterpenoid compounds. More preferably, the saponin is Quil A or the extract disclosed in Australian application no. 10777/95, or compounds obtainable from these extracts. Still more preferably, the saponin is Quil A.
As used throughout the description and claims the term xe2x80x9cpolycationic polyelectrolytexe2x80x9d refers to polymer or oligomers, natural or synthetic, that, by virtue of their chemical structure, acquire a plurality of discrete positive charges in aqueous solution under appropriate pH conditions. Suitable polycationic polyelectrolytes are DEAB dextran, polyethyleneimine, ethoxylated polyethyleneimine, epichlorhydrin-modified polyethyleneimine, diethylaminoethyl ester and amide derivatives of acrylate polymers, copolymers and the like. The most preferable polycationic polyelectrolyte is DEAE dextran, which is a polycationic derivative of dextran (average molecular weight 10000 to 1000000, preferably 200000 to 750000, most preferably 500000) containing diethylamino ethyl groups linked to glucose in a 1:3 ratio. Typically the polycationic polyelectrolyte is in aqueous solution, for example, phosphate buffered saline.
While the invention embraces a wide range of immunoadjuvant oils, mineral oils are preferred. More preferred are those mineral oils already known in the art for use as adjuvants and including substances such as Drakeol, Markol, squalene, squalane and the like but the preferred mineral oil is Montanide oil. Mineral oil immunoadjuvants are frequently referred to as Freund""s incomplete adjuvant and this adjuvant typically comprises 85% mineral oil and 15% mannide monooleate as an emulsifier.
Typically the adjuvant composition of the present invention takes the form of an emulsion with the polycationic polyelectrolyte dissolved in the aqueous phase and the mineral oil forming the non-aqueous phase. It is well known that immunoadjuvant emulsions of individual oils used separately can be formulated with oil to water phase ratios extending over a broad range and embracing the ratios 80:20 to 20:80 (v/v) for example, more preferably 60:40 to 40:60 (v/v). Such a broad range of ratios of oil phase to aqueous phase also applies in the present invention except that the aqueous phase will always comprise a polycationic polyelectrolyte solution and the composition will also include a saponin. While not wishing to be bound by theory, the saponin is amphiphilic and which may partition itself between the phases with the hydrophilic sugar residues in the aqueous phase and the hydrophobic triterpenoid structure in the non-aqueous phase. Accordingly, the saponin may serve to stabilise the emulsion.
Preferably, vaccines including adjuvant compositions in accordance with the present invention contain the saponin component at a concentration greater than 50 xcexcg/ml and the polycationic polyelectrolyte at a concentration of greater than 1 mg/ml. More preferably, they contain saponins in a concentration of greater than 100 xcexcg/ml and the polycationic polyelectrolyte component in a concentration of greater than 1.5 mg/ml. The upper limits of concentration of the saponin component and the polycationic polyelectrolyte are essentially determined by economic considerations since these components are expensive, but the saponin may be present in concentrations up to 10 mg/ml, typically up to 1 mg/ml, and the polycationic polyelectrolyte may be present in concentrations up to 200 mg/ml, typically 150 mg/ml.
The emulsifiers used to form the novel compositions of the invention are those known in the art such as mannide monooleate, Arlacela A, Arlacela 80 and Tween 80. It will be recognised by those skilled in the art that the adjuvant composition can be used in virtually any vaccine including any antigenic substance, although it will be recognised that many factors other than the nature of the adjuvant composition will influence the nature of and level of the antibody response to the vaccine.
The adjuvant composition is particularly useful when used in conjunction with a whole cell killed vaccine or killed viral vaccine or a vaccine comprising a proteinaceous substance, which may or may not be glyocosylated or otherwise chemically modified, alone or as a carrier for a low molecular weight compound. In general, the antigenic substance will give rise to an immune response against a disease-causing agent but may also give rise to antibodies against an agent (such as a hormone) which does not normally give rise to a disease. The disease causing agent may be a structural component or toxin of a virus, bacteria or other microbe. Examples of virally-caused diseases which may be controlled by vaccines including the adjuvant composition of the present invention include infectious bursal disease virus, Newcastle disease, infectious bronchitis virus, pseudorabies, parvovirus, classical swine fever, equine influenza, bovine viral diarrhoea virus and canine corona virus. Examples of bacterially-caused diseases include atrophic rhinitis, loptospirosis, clostridial infections, bordetella brochisepticum infections in cats, coryza in poultry, fowl chloera, Mycoplasma gallisepticum infections in poultry, pleuropmeumonia and rabies. The adjuvant composition may also be used in conjunction with sub-unit vaccines produced using recombinant DNA technology such as in a sub-unit vaccine against cattle ticks.
The antigenic substance may also comprise a target low molecular weight compound conjugated to a carrier selected so as not to be recognised by the organism as xe2x80x9cselfxe2x80x9d and thereby to generate an immune response against the low molecular weight compound. Suitable carriers include fetuin, ovalbumin, bovine serum albumin, foetal calf serum and human serum albumin. Alternatively, the carrier may be keyhole limpet haemocyanin or beta-galactosidase, among others. The low molecular weight compound may be conjugated to the carrier by any convenient means. Suitable conjugators include glutaraldehyde, toluene diisocyanate, carbodiimide, or any other suitable conjugator.
The small molecules which may be conjugated to a character include toxins such as phomopsin or other substances such as mammaliam hormones or steroids against which it may be desirable to raise an immune response.
Other antigens which may be employed include red blood cells and virus like particles, particularly VLP/NS2.
Preferably, the antigenic substance is a fetuin-phomopsin conjugate, phomopsin A conjugated to ovalbumin, phomopsin A-fetal calf serum conjugate, a virus-like particle, particularly VLP/NS2 (a VLP comprising a blue tongue virus antigen encoded by a recombinant baculovirus vector), sheep red blood cells, or ovalbumin.
In the method of the invention the antigenic substance and adjuvant composition are conveniently mixed prior to administration. Typically, the antigenic substance is in aqueous solution, such as phosphate-buffered saline. The polycationic polyelectrolyte may also be in aqueous solution, such as in solution in phosphate-buffered saline, and together these components form the aqueous phase of the emulsion. However, it will be appreciated that the antigenic substance and adjuvant may be administered sequentially, and even that the various components of the adjuvant composition may be administered sequentially rather than simultaneously provided that they undergo a physiological interaction in vivo.
Throughout the specification, except where the context requires otherwise due to express language or necessary implication, the word xe2x80x9ccomprisingxe2x80x9d is used in the sense of xe2x80x9cincludingxe2x80x9d, ie. the features specified may be associated with further features in various embodiments of the invention and are not to be construed, necessarily, as the only features of the invention.